The present invention relates to a biological material comprising mesenchymal stem cells and a three-dimensional matrix comprising a hyaluronic acid derivative thereof and the use of said biological material optionally in association with therapeutically effective ingredients and/or pharmaceutically acceptable excipients and/or diluents for the repair of connective tissue defects.
The loss of connective tissue and in particular cutaneous material due to various causes, traumatic, or metabolic, for example, can sometimes prove to be very slow healing.
This can be due to metabolic or local circulatory causes, the patient""s poor state of health or to the size of the wound, as in the case of extensive burns. The ineffectiveness of pharmacological therapy has led physicians to resort to reconstructive surgery, using connective tissue graft, such as skin graft, from the same patients whenever possible. An important breakthrough in the treatment of such lesions is the use of techniques for in vitro cell cultures.
J. Rheinwald and H. Green (Cell, 6,1975, 331-344) were the first to isolate keratinocytes which could be successfully used to cover skin lesions in clinical practice (G. G. Gallico et al., N. Engl., J Med., 311, (1984), 488-451). This innovative technique proved to have its limits, however, the most serious being the extreme fragility of the epithelial layer and the very low take rate. To overcome these limitations, dermal derivatives have been reconstructed on which keratynocytes can be grown. Yannas et al (Science, 215 (1982), 174-176) used a mixture of collagen and glycosaminoglycans to obtain a porous reabsorbable material to serve as a skin substitute on lesions characterised by the loss of cutaneous substance.
S. Boyce and J. Hansbrough (Surgery, 103 (1988), 421-431) described the use of layers of collagen and glycosaminoglycans as supports on which to grow keratinocytes for subsequent graft.
Another system for the preparation of dermal substitutes is represented by fibroblast cultures on biocompatible three-dimensional matrices based on synthetic or semisynthetic polymers. It is possible to seed and grow fibroblasts on these structures, thus enabling the production of an extracellular matrix similar to that of natural connective tissue.
Some well-known examples of dermal substitutes are:
1. Dermagraft, developed by Advanced Tissue Science (California), in which human fibroblasts are seeded and cultivated on a matrix formed by polylactic, polyglycolic or polygalactoside acid. These fibroblast-populated matrices are subsequently seeded with keratinocytes, to enhance their more xe2x80x9cphysiologicalxe2x80x9d growth;
2. Graft-skin by Organogenesis Inc. (Boston USA) composed of a collagen substrate on which heterologous human fibroblasts are seeded;
3. Alloderm, produced by Life Cell Corp.(Texas USA) constituted by human or pig dermis, left intact and stored at a low temperature. Before use it can be seeded with autologous fibroblasts and keratinocytes and then used for grafting.
Although these products represent good biological supports for in vitro cultures, their in vivo application is somewhat limited, due to immunological reactions against their non autologous components, as well as to the risk of viral contamination.
Another problem involved in the preparation of connective tissue substitutes is represented by the supply of connective tissue cells to seed onto the biocompatible matrices. Indeed it is not always easy to isolate connective tissue cells, especially in the case of elderly or severely weakened subjects. One solution to this problem is offered by mesenchymal stem cells. Mesenchymal stem cells (MSC) are the formative pluripotential blast cells found inter alia in bone marrow, blood dermis and periosteum that are capable of differentiating into any of the specific types of mesenchymal stem or connective tissue cells such as adipose, osseous, cartilaginous, elastic, and fibrous connective tissues, depending upon various influences from bioactive factors such as cytokines. Although these cells are normally present at very low frequencies in bone marrow, a process has been discovered for isolating, plurifying and greatly replicating these cells in cultures, as disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359.
In order to isolate human MSC, it is necessary to isolate rare pluripotent mesenchymal stem cells from other cells in the bone marrow or other MSC source. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary spaces. Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, blood and other mesenchymal stem cell tissues.
Isolated human mesenchymal stem cell compositions serve as the progenitors for various mesenchymal cell lineages. Isolated mesenchymal stem cell populations have the ability to expand in culture without differentiating and have the ability to differentiate into specific mesenchymal lineages when either induced in vitro or placed in vivo at the place of the damaged tissue.
Hyaluronic acid or hyaluronan is a linear polyanionic polysaccharide, a member of the family known as glycosaminoglycans. It is present in most vertebrate connective tissue at relatively high concentrations (up to 10 mg/ml) (Kvam, K. C. Granese, D. Flaibani, A., Zanetti, F. and Paoletti, S (1993) Anal. Biochem. 211, 44-49).
The basic structural unit is a disaccharide consisting of D-glucuronic acid (GIcA) in b1-3 linkage to N-acetyl-D-glucosamine (GIcNAc) and the disaccharides are linked together in a b1-4 linkage. The molecular weight may be as high as 1xc3x97107 Da, so that in its highly hydrated form, hyaluronan shows unique properties of viscoelasticity and plasticity.
In cartilage, hyaluronic acid plays a central role in the assembly of the macromolecular components that constitute the extracellular matrix (Hardingham T. E. and Muir, H (1972) Biochim. Biophys. Acta 279,401-405) It binds with high specificity and affinity to aggregan and link protein (Neame, P. J, and Barry F. P. (1994) Experientia 49, 393-402.). A single hyaluronic acid chain may form a central xe2x80x9cfilamentxe2x80x9d that binds a large number of aggregan molecules, forming a supermolecular complex that immobilises water and leads to a highly hydrated gel-like structure (Morgelin, M., Paulson, M., and Angel J., (1990) Biochem. Soc. Trans. 18, 204-207). In addition hyaluronic acid binds with high affinity to the chondrocyte receptor CD44 (Knudson, C. B. (1993) J. Cell Biochem. 120, 825-834). Hyaluronic acid is present in the vitreous of the eye and in the synovial fluid in joint cavities. It is used in surgical procedures involving the anterior portion of the eye, such as corneal transplants and the removal and replacement of a cataractous lens.(Balasz E. A. (1991) xe2x80x9cCosmetic and pharmaceutical Applications of polymersxe2x80x9d Gebelein C. G. et al ed.) Plenum Press New York). It is also used in the therapy of arthritis where injection of hyaluronic acid into the joint space may restore the rheological properties of the synovial fluid.(Larsen, N. E., Lombard, K. M., Parent, E. G. and Balasz, E. A., (1992) J. Orthop. Res. 10, 23-32).
Hyaluronic acid can be chemically treated to alter its biophysical and biological properties. For instance, it can be treated with formaldehyde or vinyl sulfone to give rise to cross-linked gels which have rheological properties different from the native molecule. (Abatangelo, G., Brun P., and Cortivo R.(1994) xe2x80x9cNovel biomaterials based on hyaluronic acid and its derivativesxe2x80x9d(Williams D. F. Ed) Fidia Advanced Biopolymers S.r.l. Italy). In addition, various hyaluronic acid derivatives are known from literature, for example the hyaluronic acid esters with different aromatic aliphatic and/or araliphatic alcohols like those disclosed in U.S. Pat. No. 4,851,521, with altered rheological properties if compared with the hyaluronic acid (Benedetti L., Bellini, D., Renier, D and O""Reagan M, (1994) in xe2x80x9cNovel biomaterials based on hyaluronic acid and its derivativesxe2x80x9d (Williams D. F. Ed) Fidia Advanced Biopolymers s.r.l. Italy). The water solubility of hyaluronic acid is dramatically reduced in the above mentioned hyaluronic acid esters, thus resulting in a major increase in the biological performance of this material (the previous reference).
Hyaluronic acid esters are known to be used in skin grafts thanks to their highly biocompatible and biodegradable materials. (Benedetti et al., Biomaterials, 14 (1993) 1154-1160; R. Cortivo et al., Biomaterials, 12 (1991) 727-730) and their lack of immunoreactivity.
Other hyaluronic acid derivatives such as the crosslinked hyaluronic acid polymers (ACP), namely the internal esters between the carboxylic function of a disaccharide unit of hyaluronic acid with a hydroxy function of another disaccharide unit of the same hyaluronic acid molecule or of a different hyaluronic acid molecule, are disclosed in U.S. Pat. No. 5,676,964. Both these hyaluronic acid derivatives can be in different physical forms Such as in the form of nonwoven tissue, sponges, gels etc.
In one aspect the present invention relates to a biological material comprising the following components:
a) a cell preparation enriched in mesenchymal stem cells,
b) a three-dimensional matrix containing a hyaluronic acid derivative.
In another aspect the present invention relates to a composition comprising the above biologic material in combination with pharmaceutically acceptable excipients and/or diluents possibly associated with therapeutically active ingredients, for the repair of connective tissue defects.
In an other aspect the present invention relates to implants consisting essentially of said biological material, for the repair of connective tissue defects
In another aspect, the present invention provides a therapeutic method for de novo formation of connective tissue in vivo by introducing into a site for de novo connective tissue formation in an individual in need thereof a therapeutically effective amount of the above mentioned composition comprising the biological material according to the present invention.
Finally, the present invention relates also to a surgical method for de novo formation of connective tissue in vivo by introducing into a site for de novo connective tissue formation in an individual in need thereof the above mentioned implants consisting essentially of the biological material, according to the present invention.